Hexamethonium‐ and methyllycaconitine‐induced changes in acetylcholine release from rat motor nerve terminals

Abstract

1 The neuronal nicotinic receptor antagonists hexamethonium and methyllycaconitine (MLA) have been used to study the putative prejunctional nicotinic ACh receptors (AChRs) mediating a negativefeedback control of ACh release from motor nerve terminals in voltage-clamped rat phrenic nerve/ hemidiaphragm preparations. 2 Hexamethonium (200 mM), but not MLA (0.4 ± 2.0 mM), decreased the time constant of decay of both endplate currents (e.p.cs) and miniature endplate currents (m.e.p.cs), indicating endplate ion channel block with hexamethonium. However, driving function analysis and reconvolution of e.p.cs and m.e.p.cs indicated that this ion channel block did not compromise the analysis of e.p.c. quantal content. 3 At low frequencies of stimulation (0.5 ± 2 Hz), hexamethonium (200 mM) and MLA (2.0 mM) increased e.p.c. quantal content by 30 ± 40%. At high frequencies (50 ± 150 Hz) neither compound a ected e.p.c. quantal content. All e ects on quantal content were paralleled by changes in the size of the pool of quanta available for release. 4 The low frequency augmentation of e.p.c. quantal content by hexamethonium was absent when extracellular [Ca 2+ ] was lowered from 2.0 to 0.5 mM. 5 At the concentrations studied, MLA and hexamethonium produced a small (10 ± 20%) decrease in the peak amplitude of m.e.p.cs. 6 Neither apamin (100 nM) nor charybdotoxin (80 nM) had e ects on spontaneous or nerve evoked current amplitudes at any frequency of stimulation. Thus the ability of nicotinic antagonists to augment e.p.c. quantal content is not due to inhibition of Ca 2+ -activated K + -channels. 7 We suggest that hexamethonium and MLA increase evoked ACh release by blocking prejunctional nicotinic AChRs. These receptors exert a negative feedback control over evoked ACh release and are probably of the a-bungarotoxin-insensitive neuronal type.

Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction

“…These DMPP‐induced reductions probably reflect the appearance of a small degree of endplate ion channel block. However, given that the reductions in time constants of decay were small and similar for both MEPCs and EPCs, we consider it unlikely that the presence of this endplate ion channel block would have a significant impact on the quantal analysis of ACh release (see also Tian et al 1997).…”

“…Firstly, effects of channel block on current amplitudes are only detected in association with large decreases in the decay time constants of the signals (Prior et al 1990), something not seen here. Secondly, driving function analysis has shown that, under the conditions employed here, small amounts of endplate ion channel block equally affect MEPC and EPC amplitudes and so do not affect the analysis of ACh release (Tian et al 1997). Finally, the accumulation of DMPP‐blocked endplate channels during repetitive 50 Hz stimulation cannot underlie the differential effects of the compound on EPC and MEPC amplitudes since this would be expected to depress EPCs more than MEPCs and hence produce an apparent decrease in evoked ACh release.…”

“…Because of an initial rundown of EPC amplitude at the start of the 50 Hz stimulation period only the last eighty signals, where EPC amplitude was constant, were considered for analysis. For MEPCs we have determined that the best estimates of peak amplitude and time course are obtained from averaged current records (Tian et al 1994, 1997). Therefore, for each experimental condition 40–200 individual MEPCs were averaged, following alignment to the midpoint of their rising phases, and the single average signal was subsequently analysed in the same manner as the individual EPCs.…”

“…hexamethonium) produce a Ca 2+ ‐dependent increase in ACh release at low frequencies of nerve stimulation (Wilson & Thomsen, 1992; Tian et al 1997). The complex effects of nicotinic ACh receptor antagonists on ACh release have been interpreted in terms of multiple facilitatory and inhibitory subclasses of prejunctional nicotinic ACh autoreceptors (Tian et al 1994, 1997). However, to date, no validation of the model has been made with respect to the prejunctional actions of nicotinic ACh receptor agonists.…”

“…Thus, the study strengthens the hypothesis that there exists, on rat motor terminals, muscle‐type nicotinic ACh receptors, activation of which leads to an enhancement of its release during periods of sustained motor terminal activity. Further, the ability of W7 to attenuate the prejunctional actions of DMPP and vecuronium suggests a role for calmodulin in the facilitatory effects of ACh hypothesized to be mediated by prejunctional positive‐feedback muscle‐type nicotinic ACh receptors (Tian et al 1994, 1997).…”